Neural differentiation of mouse embryonic stem cells on conductive nanofiber scaffolds

Nerve tissue engineering requires suitable precursor cells as well as the necessary biochemical and physical cues to guide neurite extension and tissue development. An ideal scaffold for neural regeneration would be both fibrous and electrically conductive. We have contrasted the growth and neural differentiation of mouse embryonic stem cells on three different aligned nanofiber scaffolds composed of poly L: -lactic acid supplemented with either single- or multi-walled carbon-nanotubes. The addition of the nanotubes conferred conductivity to the nanofibers and promoted mESC neural differentiation as evidenced by an increased mature neuronal markers expression. We propose that the conductive scaffold could be a useful tool for the generation of neural tissue mimics in vitro and potentially as a scaffold for the repair of neural defects in vivo.

Predicting the Timing of Spikes Evoked by Tactile Stimulation of the Hand

Kim SS, Sripati AP, Bensmaia SJ. Predicting the timing of spikes evoked by tactile stimulation of the hand. J Neurophysiol 104: 1484-1496, 2010. First published July 7, 2010; doi: 10.1152/jn.00187.2010. What does the hand tell the brain? Tactile stimulation of the hand evokes remarkably precise patterns of neural activity, suggesting that the timing of individual spikes may convey information. However, many aspects of the transformation of mechanical deformations of the skin into spike trains remain unknown. Here we describe an integrate-and-fire model that accurately predicts the timing of individual spikes evoked by arbitrary mechanical vibrations in three types of mechanoreceptive afferent fibers that innervate the hand. The model accounts for most known properties of the three fiber types, including rectification, frequency-sensitivity, and patterns of spike entrainment as a function of stimulus frequency. These results not only shed light on the mechanisms of mechanotransduction but can be used to provide realistic tactile feedback in upper-limb neuroprostheses.

Diacylglycerol Kinase Is Stimulated by Arachidonic Acid in Neural Membranes.

The effect of arachidonic acid (AA) on the activity of diacylglycerol (DG) kinase in neural membranes was investigated. When rat brain cortical membranes were incubated with 0.5 mM dipalmitin and [gamma-P-32]ATP, formation of phosphatidic acid (PA) was observed. It was linear up to 5 min, and the initial rate was similar to 1.0 nmol/min/mg of protein. The DG kinase activity was stimulated twofold by 0.25 mM AA. The stimulation was apparent at the earliest time point measured (1 min) and with the lowest concentration of AA tested (62.5 mu M). The stimulation was proportional to the concentration of AA up to 250 mu M. AA was the most potent stimulator of DG kinase, and linolenic acid showed similar to 40% stimulation. Oleic acid showed no effect, whereas linoleic and the saturated fatty acids tested were inhibitory. AA stimulation of DG kinase was observed only with membranes of cerebrum, cerebellum, and myelin and not with brain cytosol or liver membranes. AA also stimulated the formation of PA in the absence of added dipalmitin (endogenous activity) with membranes prepared from whole brain. DG kinase of neural membranes was extracted with 2 M NaCl, which on dialysis yielded a precipitate. Both the precipitate and the supernatant showed DG kinase activity, but only the enzyme in the precipitate was stimulated by AA at concentrations as low as 25 mu M. It is suggested that AA, through its effect on DG kinase, regulates the level of DG in neural membranes, which in turn regulates protein kinase C activity.

Vertical electric field stimulated neural cell functionality on porous amorphous carbon electrodes

We demonstrate the efficacy of amorphous macroporous carbon substrates as electrodes to support neuronal cell proliferation and differentiation in electric field mediated culture conditions. The electric field was applied perpendicular to carbon substrate electrode, while growing mouse neuroblastoma (N2a) cells in vitro. The placement of the second electrode outside of the cell culture medium allows the investigation of cell response to electric field without the concurrent complexities of submerged electrodes such as potentially toxic electrode reactions, electro-kinetic flows and charge transfer (electrical current) in the cell medium. The macroporous carbon electrodes are uniquely characterized by a higher specific charge storage capacity (0.2 mC/cm(2)) and low impedance (3.3 k Omega at 1 kHz). The optimal window of electric field stimulation for better cell viability and neurite outgrowth is established. When a uniform or a gradient electric field was applied perpendicular to the amorphous carbon substrate, it was found that the N2a cell viability and neurite length were higher at low electric field strengths (<= 2.5 V/cm) compared to that measured without an applied field (0 V/cm). While the cell viability was assessed by two complementary biochemical assays (MTT and LDH), the differentiation was studied by indirect immunostaining. Overall, the results of the present study unambiguously establish the uniform/gradient vertical electric field based culture protocol to either enhance or to restrict neurite outgrowth respectively at lower or higher field strengths, when neuroblastoma cells are cultured on porous glassy carbon electrodes having a desired combination of electrochemical properties. (C) 2013 Elsevier Ltd. All rights reserved.

Intermittent electrical stimuli for guidance of human mesenchymal stem cell lineage commitment towards neural-like cells on electroconductive substrates

In the context of the role of multiple physical factors in dictating stem cell fate, the present paper demonstrates the effectiveness of the intermittently delivered external electric field stimulation towards switching the stem cell fate to specific lineage, when cultured in the absence of biochemical growth factors. In particular, our findings present the ability of human mesenchymal stem cells (hMSCs) to respond to the electric stimuli by adopting extended neural-like morphology on conducting polymeric substrates. Polyaniline (PANI) is selected as the model system to demonstrate this effect, as the electrical conductivity of the polymeric substrates can be systematically tailored over a broad range (10(-9) to 10 S/cm) from highly insulating to conducting by doping with varying concentrations (10(-5) to 1 M) of HCl. On the basis of the culture protocol involving the systematic delivery of intermittent electric field (dc) stimulation, the parametric window of substrate conductivity and electric field strength was established to promote significant morphological extensions, with minimal cellular damage. A time dependent morphological change in hMSCs with significant filopodial elongation was observed after 7 days of electrically stimulated culture. Concomitant with morphological changes, a commensurate increase in the expression of neural lineage commitment markers such as nestin and PI tubulin was recorded from hMSCs grown on highly conducting substrates, as revealed from the mRNA expression analysis using Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) as well as by immune-fluorescence imaging. Therefore, the present work establishes the key role of intermittent and systematic delivery of electric stimuli as guidance cues in promoting neural-like differentiation of hMSCs, when grown on electroconductive substrates. (C) 2014 Elsevier Ltd. All rights reserved.

Stimulation discomfort comparison of asynchronous and synchronous methods with multi-field electrodes

Functional Electrical Stimulation (FES) is a technique that consists on applying electrical current pulses to artificially activate motor nerve fibers and produce muscle contractions to achieve functional movements. The main applications of FES are within the rehabilitation field, in which this technique is used to aid recovery or to restore lost motor functions. People that benefit of FES are usually patients with neurological disorders which result in motor dysfunctions; most common patients include stroke and spinal cord injury (SCI). Neuroprosthesis are devices that have their basis in FES technique, and their aim is to bridge interrupted or damaged neural paths between the brain and upper or lower limbs. One of the aims of neuroprosthesis is to artificially generate muscle contractions that produce functional movements, and therefore, assist impaired people by making them able to perform activities of daily living (ADL). FES applies current pulses and stimulates nerve fibers by means of electrodes, which can be either implanted or surface electrodes. Both of them have advantages and disadvantages. Implanted electrodes need open surgery to place them next to the nerve root, so these electrodes carry many disadvantages that are produced by the use of invasive techniques. In return, as the electrodes are attached to the nerve, they make it easier to achieve selective functional movements. On the contrary, surface electrodes are not invasive and are easily attached or detached on the skin. Main disadvantages of surface electrodes are the difficulty of selectively stimulating nerve fibers and uncomfortable feeling perceived by users due to sensory nerves located in the skin. Electrical stimulation surface electrode technology has improved significantly through the years and recently, multi-field electrodes have been suggested. This multi-field or matrix electrode approach brings many advantages to FES; among them it is the possibility of easily applying different stimulation methods and techniques. The main goal of this thesis is therefore, to test two stimulation methods, which are asynchronous and synchronous stimulation, in the upper limb with multi-field electrodes. To this end, a purpose-built wrist torque measuring system and a graphic user interface were developed to measure wrist torque produced with each of the methods and to efficiently carry out the experiments. Then, both methods were tested on 15 healthy subjects and sensitivity results were analyzed for different cases. Results show that there are significant differences between methods regarding sensation in some cases, which can affect effectiveness or success of FES.

Psychobiophysics of transcranial magnetic stimulation

Transcranial magnetic stimulation (TMS) is a technique that stimulates the brain using a magnetic coil placed on the scalp. Since it is applicable to humans non-invasively, directly interfering with neural electrical activity, it is potentially a good tool to study the direct relationship between perceptual experience and neural activity. However, it has been difficult to produce a clear perceptible phenomenon with TMS of sensory areas, especially using a single magnetic pulse. Also, the biophysical mechanisms of magnetic stimulation of single neurons have been poorly understood.

In the psychophysical part of this thesis, perceptual phenomena induced by TMS of the human visual cortex are demonstrated as results of the interactions with visual inputs. We first introduce a method to create a hole, or a scotoma, in a flashed, large-field visual pattern using single-pulse TMS. Spatial aspects of the interactions are explored using the distortion effect of the scotoma depending on the visual pattern, which can be luminance-defined or illusory. Its similarity to the distortion of afterimages is also discussed. Temporal interactions are demonstrated in the filling-in of the scotoma with temporally adjacent visual features, as well as in the effective suppression of transient visual features. Also, paired-pulse TMS is shown to lead to different brightness modulations in transient and sustained visual stimuli.

In the biophysical part, we first develop a biophysical theory to simulate the effect of magnetic stimulation on arbitrary neuronal structure. Computer simulations are performed on cortical neuron models with realistic structure and channels, combined with the current injection that simulates magnetic stimulation. The simulation results account for general and basic characteristics of the macroscopic effects of TMS including our psychophysical findings, such as a long inhibitory effect, dependence on the background activity, and dependence on the direction of the induced electric field.

The perceptual effects and the cortical neuron model presented here provide foundations for the study of the relationship between perception and neural activity. Further insights would be obtained from extension of our model to neuronal networks and psychophysical studies based on predictions of the biophysical model.

Dissecting neural circuits for vision in nonhuman primates using fMRI-guided electrophysiology and optogenetics

The visual system is a remarkable platform that evolved to solve difficult computational problems such as detection, recognition, and classification of objects. Of great interest is the face-processing network, a sub-system buried deep in the temporal lobe, dedicated for analyzing specific type of objects (faces). In this thesis, I focus on the problem of face detection by the face-processing network. Insights obtained from years of developing computer-vision algorithms to solve this task have suggested that it may be efficiently and effectively solved by detection and integration of local contrast features. Does the brain use a similar strategy? To answer this question, I embark on a journey that takes me through the development and optimization of dedicated tools for targeting and perturbing deep brain structures. Data collected using MR-guided electrophysiology in early face-processing regions was found to have strong selectivity for contrast features, similar to ones used by artificial systems. While individual cells were tuned for only a small subset of features, the population as a whole encoded the full spectrum of features that are predictive to the presence of a face in an image. Together with additional evidence, my results suggest a possible computational mechanism for face detection in early face processing regions. To move from correlation to causation, I focus on adopting an emergent technology for perturbing brain activity using light: optogenetics. While this technique has the potential to overcome problems associated with the de-facto way of brain stimulation (electrical microstimulation), many open questions remain about its applicability and effectiveness for perturbing the non-human primate (NHP) brain. In a set of experiments, I use viral vectors to deliver genetically encoded optogenetic constructs to the frontal eye field and faceselective regions in NHP and examine their effects side-by-side with electrical microstimulation to assess their effectiveness in perturbing neural activity as well as behavior. Results suggest that cells are robustly and strongly modulated upon light delivery and that such perturbation can modulate and even initiate motor behavior, thus, paving the way for future explorations that may apply these tools to study connectivity and information flow in the face processing network.

Microelectrode Implants for Spinal Cord Stimulation in Rats

Paralysis is a debilitating condition afflicting millions of people across the globe, and is particularly deleterious to quality of life when motor function of the legs is severely impaired or completely absent. Fortunately, spinal cord stimulation has shown great potential for improving motor function after spinal cord injury and other pathological conditions. Many animal studies have shown stimulation of the neural networks in the spinal cord can improve motor ability so dramatically that the animals can even stand and step after a complete spinal cord transaction.

This thesis presents work to successfully provide a chronically implantable device for rats that greatly enhances the ability to control the site of spinal cord stimulation. This is achieved through the use of a parylene-C based microelectrode array, which enables a density of stimulation sites unattainable with conventional wire electrodes. While many microelectrode devices have been proposed in the past, the spinal cord is a particularly challenging environment due to the bending and movement it undergoes in a live animal. The developed microelectrode array is the first to have been implanted in vivo while retaining functionality for over a month. In doing so, different neural pathways can be selectively activated to facilitate standing and stepping in spinalized rats using various electrode combinations, and important differences in responses are observed.

An engineering challenge for the usability of any high density electrode array is connecting the numerous electrodes to a stimulation source. This thesis develops several technologies to address this challenge, beginning with a fully passive implant that uses one wire per electrode to connect to an external stimulation source. The number of wires passing through the body and the skin proved to be a hazard for the health of the animal, so a multiplexed implant was devised in which active electronics reduce the number of wires. Finally, a fully wireless implant was developed. As these implants are tested in vivo, encapsulation is of critical importance to retain functionality in a chronic experiment, especially for the active implants, and it was achieved without the use of costly ceramic or metallic hermetic packaging. Active implants were built that retained functionality 8 weeks after implantation, and achieved stepping in spinalized rats after just 8-10 days, which is far sooner than wire-based electrical stimulation has achieved in prior work.

Neural mechanisms of context effects on face recognition: automatic binding and context shift decrements.

Although people do not normally try to remember associations between faces and physical contexts, these associations are established automatically, as indicated by the difficulty of recognizing familiar faces in different contexts ("butcher-on-the-bus" phenomenon). The present fMRI study investigated the automatic binding of faces and scenes. In the face-face (F-F) condition, faces were presented alone during both encoding and retrieval, whereas in the face/scene-face (FS-F) condition, they were presented overlaid on scenes during encoding but alone during retrieval (context change). Although participants were instructed to focus only on the faces during both encoding and retrieval, recognition performance was worse in the FS-F than in the F-F condition ("context shift decrement" [CSD]), confirming automatic face-scene binding during encoding. This binding was mediated by the hippocampus as indicated by greater subsequent memory effects (remembered > forgotten) in this region for the FS-F than the F-F condition. Scene memory was mediated by right parahippocampal cortex, which was reactivated during successful retrieval when the faces were associated with a scene during encoding (FS-F condition). Analyses using the CSD as a regressor yielded a clear hemispheric asymmetry in medial temporal lobe activity during encoding: Left hippocampal and parahippocampal activity was associated with a smaller CSD, indicating more flexible memory representations immune to context changes, whereas right hippocampal/rhinal activity was associated with a larger CSD, indicating less flexible representations sensitive to context change. Taken together, the results clarify the neural mechanisms of context effects on face recognition.

A neural biomarker of psychological vulnerability to future life stress.

We all experience a host of common life stressors such as the death of a family member, medical illness, and financial uncertainty. While most of us are resilient to such stressors, continuing to function normally, for a subset of individuals, experiencing these stressors increases the likelihood of developing treatment-resistant, chronic psychological problems, including depression and anxiety. It is thus paramount to identify predictive markers of risk, particularly those reflecting fundamental biological processes that can be targets for intervention and prevention. Using data from a longitudinal study of 340 healthy young adults, we demonstrate that individual differences in threat-related amygdala reactivity predict psychological vulnerability to life stress occurring as much as 1 to 4 years later. These results highlight a readily assayed biomarker, threat-related amygdala reactivity, which predicts psychological vulnerability to commonly experienced stressors and represents a discrete target for intervention and prevention.

Age-related effects on the neural correlates of autobiographical memory retrieval.

Older adults recall less episodically rich autobiographical memories (AM), however, the neural basis of this effect is not clear. Using functional MRI, we examined the effects of age during search and elaboration phases of AM retrieval. Our results suggest that the age-related attenuation in the episodic richness of AMs is associated with difficulty in the strategic retrieval processes underlying recovery of information during elaboration. First, age effects on AM activity were more pronounced during elaboration than search, with older adults showing less sustained recruitment of the hippocampus and ventrolateral prefrontal cortex (VLPFC) for less episodically rich AMs. Second, there was an age-related reduction in the modulation of top-down coupling of the VLPFC on the hippocampus for episodically rich AMs. In sum, the present study shows that changes in the sustained response and coupling of the hippocampus and prefrontal cortex (PFC) underlie age-related reductions in episodic richness of the personal past.

Imagery and retrieval of auditory and visual information: neural correlates of successful and unsuccessful performance.

Remembering past events - or episodic retrieval - consists of several components. There is evidence that mental imagery plays an important role in retrieval and that the brain regions supporting imagery overlap with those supporting retrieval. An open issue is to what extent these regions support successful vs. unsuccessful imagery and retrieval processes. Previous studies that examined regional overlap between imagery and retrieval used uncontrolled memory conditions, such as autobiographical memory tasks, that cannot distinguish between successful and unsuccessful retrieval. A second issue is that fMRI studies that compared imagery and retrieval have used modality-aspecific cues that are likely to activate auditory and visual processing regions simultaneously. Thus, it is not clear to what extent identified brain regions support modality-specific or modality-independent imagery and retrieval processes. In the current fMRI study, we addressed this issue by comparing imagery to retrieval under controlled memory conditions in both auditory and visual modalities. We also obtained subjective measures of imagery quality allowing us to dissociate regions contributing to successful vs. unsuccessful imagery. Results indicated that auditory and visual regions contribute both to imagery and retrieval in a modality-specific fashion. In addition, we identified four sets of brain regions with distinct patterns of activity that contributed to imagery and retrieval in a modality-independent fashion. The first set of regions, including hippocampus, posterior cingulate cortex, medial prefrontal cortex and angular gyrus, showed a pattern common to imagery/retrieval and consistent with successful performance regardless of task. The second set of regions, including dorsal precuneus, anterior cingulate and dorsolateral prefrontal cortex, also showed a pattern common to imagery and retrieval, but consistent with unsuccessful performance during both tasks. Third, left ventrolateral prefrontal cortex showed an interaction between task and performance and was associated with successful imagery but unsuccessful retrieval. Finally, the fourth set of regions, including ventral precuneus, midcingulate cortex and supramarginal gyrus, showed the opposite interaction, supporting unsuccessful imagery, but successful retrieval performance. Results are discussed in relation to reconstructive, attentional, semantic memory, and working memory processes. This is the first study to separate the neural correlates of successful and unsuccessful performance for both imagery and retrieval and for both auditory and visual modalities.

The short and long of it: neural correlates of temporal-order memory for autobiographical events.

Previous functional neuroimaging studies of temporal-order memory have investigated memory for laboratory stimuli that are causally unrelated and poor in sensory detail. In contrast, the present functional magnetic resonance imaging (fMRI) study investigated temporal-order memory for autobiographical events that were causally interconnected and rich in sensory detail. Participants took photographs at many campus locations over a period of several hours, and the following day they were scanned while making temporal-order judgments to pairs of photographs from different locations. By manipulating the temporal lag between the two locations in each trial, we compared the neural correlates associated with reconstruction processes, which we hypothesized depended on recollection and contribute mainly to short lags, and distance processes, which we hypothesized to depend on familiarity and contribute mainly to longer lags. Consistent with our hypotheses, parametric fMRI analyses linked shorter lags to activations in regions previously associated with recollection (left prefrontal, parahippocampal, precuneus, and visual cortices), and longer lags with regions previously associated with familiarity (right prefrontal cortex). The hemispheric asymmetry in prefrontal cortex activity fits very well with evidence and theories regarding the contributions of the left versus right prefrontal cortex to memory (recollection vs. familiarity processes) and cognition (systematic vs. heuristic processes). In sum, using a novel photo-paradigm, this study provided the first evidence regarding the neural correlates of temporal-order for autobiographical events.

The role of stimulus salience and attentional capture across the neural hierarchy in a stop-signal task.

Inhibitory motor control is a core function of cognitive control. Evidence from diverse experimental approaches has linked this function to a mostly right-lateralized network of cortical and subcortical areas, wherein a signal from the frontal cortex to the basal ganglia is believed to trigger motor-response cancellation. Recently, however, it has been recognized that in the context of typical motor-control paradigms those processes related to actual response inhibition and those related to the attentional processing of the relevant stimuli are highly interrelated and thus difficult to distinguish. Here, we used fMRI and a modified Stop-signal task to specifically examine the role of perceptual and attentional processes triggered by the different stimuli in such tasks, thus seeking to further distinguish other cognitive processes that may precede or otherwise accompany the implementation of response inhibition. In order to establish which brain areas respond to sensory stimulation differences by rare Stop-stimuli, as well as to the associated attentional capture that these may trigger irrespective of their task-relevance, we compared brain activity evoked by Stop-trials to that evoked by Go-trials in task blocks where Stop-stimuli were to be ignored. In addition, region-of-interest analyses comparing the responses to these task-irrelevant Stop-trials, with those to typical relevant Stop-trials, identified separable activity profiles as a function of the task-relevance of the Stop-signal. While occipital areas were mostly blind to the task-relevance of Stop-stimuli, activity in temporo-parietal areas dissociated between task-irrelevant and task-relevant ones. Activity profiles in frontal areas, in turn, were activated mainly by task-relevant Stop-trials, presumably reflecting a combination of triggered top-down attentional influences and inhibitory motor-control processes.